Pest control composition and pest control method

ABSTRACT

The present invention addresses the problem of providing a composition having excellent controlling effects with respect to pests and provides a pest control composition including bacillus strain APM-1 (new strain of Bacillus, APM-1) deposited under ATCC Accession No. PTA-4838 and one or more nicotinic acetylcholine receptor agonist compound.

TECHNICAL FIELD

The present invention relates to a composition for controlling pests anda method for controlling pests.

BACKGROUND ART

New strain of Bacillus, APM-1 (deposited under ATCC Accession No.PTA-4838), has been known as an active ingredient of compositions forcontrolling pests and disclosed, for example, in Patent Document 1.Also, nicotinic acetylcholine receptor agonist compounds were known asan active ingredient of compositions for controlling pests anddisclosed, for example, in Non-Patent Document 1. There is need for amaterial which is still more effective for controlling pests.

PRIOR ART DOCUMENTS Patent Documents

-   Patent Document 1: WO 2003/055303

Non-Patent Document

-   Non-Patent Document 1: Crop Protection HANDBOOK 2014; ISBN    1-892829-26-6

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

Damage from pests is a cause of considerable loss of crop production,and there is a need to control such pests more effectively. Thus, it isan object of the present invention to provide a composition having anexcellent controlling effect against pests.

Means for Solving the Problems

The present inventors intensively studied to achieve the above objectand have found that a composition comprising Bacillus strain APM-1 whichhas been deposited under ATCC Accession No. PTA-4838 (New strain ofBacillus, APM-1) and one or more nicotinic acetylcholine receptoragonist compound has an excellent controlling effect against pests.

Thus, the present invention includes the following [1] to [9].

-   [1] A composition for controlling pests comprising Bacillus strain    APM-1 (New strain of Bacillus, APM-1) deposited under ATCC Accession    No. PTA-4838 and one or more nicotinic acetylcholine receptor    agonist compound.-   [2] The composition according to [1] wherein the nicotinic    acetylcholine receptor agonist compound is selected from the group    consisting of clothianidin, flupyradifurone, imidacloprid,    thiacloprid and thiamethoxam.-   [3] The composition according to [1] or [2] comprising the nicotinic    acetylcholine receptor agonist compound in an amount of 10⁻⁷ to    1.5×10⁷ g per 10¹⁰ cfu of Bacillus strain APM-1.-   [4] A plant seed or a vegetative propagation organ comprising    Bacillus strain APM-1 (New strain of Bacillus, APM-1) deposited    under ATCC Accession No. PTA-4838 and one or more nicotinic    acetylcholine receptor agonist compound.-   [5] The plant seed or a vegetative propagation organ according to    [4] wherein the nicotinic acetylcholine receptor agonist compound is    selected from the group consisting of clothianidin, flupyradifurone,    imidacloprid, thiacloprid and thiamethoxam.-   [6] The plant seed or vegetative propagation organ according to [4]    or [5] comprising 10⁴ to 10¹⁴ cfu of Bacillus strain APM-1 and 0.001    to 15 g of the nicotinic acetylcholine receptor agonist compound,    per 1 kg of the seed or vegetative propagation organ.-   [7] A method for controlling pests, comprising a step of applying    Bacillus strain APM-1 (New strain of Bacillus, APM-1) deposited    under ATCC Accession No. PTA-4838 and one or more nicotinic    acetylcholine receptor agonist compound to a plant or a plant    cultivation site.-   [8] The method for controlling pests according to [7] wherein the    plant is a genetically modified plant.-   [9] A method for controlling pests, comprising a step of applying    Bacillus strain APM-1 (New strain of Bacillus, APM-1) deposited    under ATCC Accession No. PTA-4838 and one or more nicotinic    acetylcholine receptor agonist compound to pests or a place where    pests are liable to inhabit.

Effect of Invention

The present invention provides an excellent composition for protectingseeds or vegetative propagation organs and plants grown therefrom frompests.

DESCRIPTION OF EMBODIMENTS

The composition for controlling pests of the present invention(hereinafter referred to as “the present composition”) contains Bacillusstrain APM-1 (New strain of Bacillus, APM-1) deposited under ATCCAccession No. PTA-4838 (hereinafter referred to as “the presentbacterial strain”) and one or more nicotinic acetylcholine receptoragonist compound (hereinafter referred to as “the present compound”).

The present bacterial strain has been disclosed in WO 2003/055303 anddeposited under the name “New strain of Bacillus, APM-1” under ATCCAccession No. PTA-4838 at ATCC (American Type Culture Collection). WO2003/055303 describes that the strain is most similar to Bucillusamyloliquefaciens. The present bacterial strain is available from ATCCand can be cultured by a known procedure. The culture may be used as itis or may be separated and concentrated using a conventional industrialtechnique, such as, not limited to, membrane separation, centrifugalseparation, or filtration separation. The fraction of the presentbacterial strain thus obtained may be used directly as it containscertain water in the present composition, or if necessary, a driedproduct obtained by a dry method, such as freeze-dry or spray drying,may be used as the present bacterial strain.

In the present composition for controlling pests, the present compoundto be used in combination with the present bacterial strain is notlimited, so long as it has an has agonistic effect on nicotinicacetylcholine receptor, but includes compounds having agonistic effecton nicotinic acetylcholine receptor, as recited on page 7 of Mode ofAction Classification Brochure Fourth Edition-December 2014, issued byThe Insecticide Resistance Action Committee, such as clothianidin,flupyradifurone, imidacloprid, thiacloprid and thiamethoxam.

Clothianidin is a known compound and has been described, e.g., on page191 in “Crop Protection HANDBOOK 2014; ISBN 1-892829-26-6”. Clothianidincan be obtained from a commercially available formulation or produced bya known method.

Flupyradifurone is a known compound and has been described, e.g., onpage 318 in “Crop Protection HANDBOOK 2014; ISBN 1-892829-26-6”.Flupyradifurone can be obtained by producing by a known method.

Imidacloprid is a known compound and has been described, e.g., on page358 in “Crop Protection HANDBOOK 2014; ISBN 1-892829-26-6”. Imidaclopridcan be obtained from a commercially available formulation or produced bya known method.

Thiacloprid is a known compound and has been described, e.g., on page568 in “Crop Protection HANDBOOK 2014; ISBN 1-892829-26-6”. Thiaclopridcan be obtained from a commercially available formulation or produced bya known method.

Thiamethoxam is a known compound and has been described, e.g., on page568 in “Crop Protection HANDBOOK 2014; ISBN 1-892829-26-6”. Thiamethoxamcan be obtained from a commercially available formulation or produced bya known method.

The present composition can be prepared typically by mixing the presentbacterial strain and the present compound, respectively, with a solidcarrier or a liquid carrier, with addition of a surfactant or otherauxiliary agents for formulation if necessary, followed by combining thepresent bacterial strain formulation and the compound formulation thusobtained. Alternatively, the present composition can be prepared bymixing the present bacterial strain with the present compound inadvance, adding a solid carrier or a liquid carrier, with addition of asurfactant or other auxiliary agents for formulation if necessary,followed by formulating into a single formulation.

Examples of the solid carrier include mineral fine powders, such askaolin clay, pyrophyllite clay, bentonite, montmorillonite, diatomaceousearth, synthetic hydrous silicon oxide, acidic clay, talc, clay,ceramic, quartz, sericite, vermiculite, pearlite, Oya stone, anthracite,limestone, coalite, and zeolite, inorganic compounds, such as sodiumchloride, carbonate, sulfate, nitrate, and urea, organic fine powders,such as rice hulls, bran, wheat flour, and peat moss. Examples of theliquid carrier include water, vegetable oil, animal oil, and mineraloil. Examples of the auxiliary substance for formulation includeanti-freezing agents, such as ethylene glycol, and propylene glycol, andthickening agents, such as carboxymethyl cellulose, and xanthan gum.

The present composition may contain the present bacterial strain in aneffective amount, for example, at least 10⁴ cfu/g, typically 10⁴ to 10¹³cfu/g, and preferably 10⁷ to 10¹² cfu/g of the present composition.

The present composition may contain the present compound in an effectiveamount, for example, typically 0.0001 to 0.90 g, preferably 0.001 to0.80 g, per 1 g of the present composition.

The present composition typically contains 10⁻⁷ to 1.5×10⁷ g, preferably10⁻⁵ to 10⁵ g, more preferably 10⁻⁴ to 10² g of the present compound per10¹⁰ cfu of the present bacterial strain.

The term “effective amount” as used herein refers to an amount of thepresent bacterial strain and the present compound that is able to exertthe controlling effect against pests.

The method of the invention for controlling pests (hereinafter referredto as “the present controlling method”) comprises a step of applying thepresent bacterial strain and one or more of the present compounds to aplant or a plant cultivation site.

Also, in the present controlling method, the present composition may beapplied to pests or a place where pests are liable to inhabit.

In the present controlling method, the present bacterial strain and thepresent compound to be used are typically those which have beenformulated and may be applied as separate formulations or as a presentcomposition. The separate formulations may be applied simultaneously orindependently.

In the present controlling method, the present bacterial strain and thepresent compound are applied in an effective amount.

In the present invention, examples of the cultivation site of the plantor the place where pests are liable to inhabit include paddy field,cultivated field, tea field, fruit orchard, non-agricultural land,seedling tray and nursery box, nursery soil and nursery mat, waterculture medium in hydroponic farm, and the like. In a cultivation siteof plant or a place where pests are liable to inhabit, pests may havealready inhabited or not yet inhabited.

In the present controlling method, examples of the method for treatingthe present bacterial strain and the present compound include foliagetreatment, soil treatment, root treatment, seed treatment and vegetativepropagation organ treatment.

Examples of the foliage treatment include treatment of the surface ofthe cultivated plant with spraying onto the foliage and stem.

Examples of the root treatment include immersing whole plant or a rootof the plant in a solution containing the present bacterial strain andthe present compound, as well as attaching a solid preparationcontaining the present bacterial strain, the present compound and asolid carrier to a root of the plant.

Examples of the soil treatment include soil broadcast, soilincorporation and chemical irrigation to soil.

Examples of the seed treatment and vegetative propagation organtreatment include applying seed treatment or vegetative propagationtreatment using the present composition, specifically, such as spraytreatment wherein a suspension of the present composition is sprayedonto the surface of the seed or the vegetative propagation organ, wetpowder coating treatment wherein the present composition in a form ofwettable powder is coated onto moist seed or vegetative propagationorgan, smearing treatment wherein a liquid of the present compositionprepared from wettable powder, emulsifiable concentrate or flowableformulation of the present composition, with addition of water ifnecessary, is applied onto seed or vegetative propagation organ,immersion treatment wherein seeds or vegetative propagation organs areimmersed in a liquid containing the present composition for a certainperiod of time, and film coating treatment and pellet coating treatmentof seeds with the present composition.

In the present invention, the simply described “plant” encompasses inits meaning “a seed of the plant” and “a vegetative propagation organ ofthe plant”.

The term “vegetative propagation organ” as used herein means a part ofroot, stem, leaf or the like of the plant having the ability to growwhen it is separated from the body and placed on soil, such as flowerbulb, potato tuberous root, stem tuber, scaly bulb, corm, rhizophore,and strawberry runner.

In the present controlling method, the amount of the present bacterialstrain and the present compound in the treatment varies depending on thekind of plant to be treated, the kind of pest to be targeted, and theoccurrence frequency, the formulation form, the treatment period, thetreatment method, the place to be treated, the weather condition or thelike, and when a stem and a leaf of the plant or a soil where the plantgrows is treated, the amount of the present bacterial strain for thetreatment is usually 10⁵ to 10¹⁹ cfu, preferably 10⁷ to 10¹⁷ cfu, per 1ha, and the amount of the present compound for the treatment is usually10 to 5000 g, preferably 20 to 2000 g, per 1 ha. The composition in aform of wettable powder, water dispersible granules or the like may beused by diluting with water so that the concentration of the presentbacterial strain is usually 10³ to 10¹² cfu/L and that the concentrationof the present compound is usually 0.0005 to 1% by weight. Thecomposition in a form of dustable powder or granules may be used as itis.

In the seed treatment or vegetative propagation organ treatment, theamount of the present bacterial strain is usually 10⁴ to 10¹⁴ cfu,preferably 10⁶ to 10¹³ cfu per 1 kg of the seed or vegetativepropagation organ, and the amount of the present compound is usually0.001 to 15 g, preferably 0.01 to 10 g, per 1 kg of the seed orvegetative propagation organ.

The weight of the seed or vegetative propagation organ means the weightthereof when treating with the present bacterial strain and the presentcompound or other agricultural chemicals before seeding or burying ofthe same.

By treating the seed or vegetative propagation organ as described above,a seed or vegetative propagation organ comprising the present bacterialstrain and one or more compounds of the invention can be obtained. Anadjuvant may be admixed if necessary during the seed treatment orvegetative propagation organs treatment.

Examples of the plant to which the present invention is applicableinclude the followings.

Agricultural crops: cereal crops, such as corn, sorghum, wheat asrepresenting wheat, barley, rye, and oat, rice, millet; pseudocereals,such as buckwheat, amaranth, quinoa; pulses, such as soybean, peanut;oilseed rape; sugar beet; cotton; sunflower; tobacco; sugar cane; hop.

Vegetables: solanaceous crops (eggplant, tomato, potato, chili pepper,green pepper, etc.), cucurbitaceae crops (cucumber, pumpkin, zucchini,watermelon, melon, orienta melon, etc.), cruciferous vegetables (radish,turnip, horseradish, kohlrabi, Chinese cabbage, cabbage, mustard,broccoli, cauliflower, etc.), asteraceae vegetables (burdock, garlandchrysanthemum, artichoke, lettuce, etc.), liliaceae vegetables (greenonion, onion, garlic, asparagus, etc.), umbelliferae vegetables (carrot,parsley, celery, parsnip, etc.), chenopodiaceae vegetables (spinach,chard, etc.), labiatae vegetables (perilla, mint, basil, etc.),leguminous crops (pea, kidney bean, adzuki bean, broad bean, chickpea,etc.), strawberry, sweet potato, yam, taro, konjac, ginger, okra.

Fruit trees: pome fruits (apple, Japanese pear, common pear, Chinesequince, quince, etc.), stone fruits (peach, plum, nectarine, Japaneseplum, cherry, apricot, prune, etc.), citrus fruits (Satsuma mandarin,orange, lemon, lime, grapefruit, etc.), nuts (chestnut, walnut, hazelnut, almond, pistachio, cashew nut, macadamia nut, etc.), berries(blueberry, cranberry, blackberry, raspberry, etc.), grape, Japanesepersimmon, olive, loquat, banana, coffee, date palm, coconut palm, oilpalm.

Trees other than fruit trees: tea, mulberry, flowering trees (azalea,camellia, hydrangea, sasanqua, Japanese star anise, cherry, tulip tree,crape myrtle, orange osmanthus, etc.), street trees (ash tree, birch,dogwood, eucalyptus, ginkgo, lilac, maple tree, oak, poplar, cercis,Chinese sweet gum, plane tree, zelkova, Japanese arborvitae, fir tree,Japanese hemlock, needle juniper, pine, spruce, yew, elm, horsechestnut, etc.), coral tree, podocarpus, cedar, Japanese cypress,croton, Japanese spindle tree, Japanese photinia.

Grasses: zoysia (zoysiagrass, Zoysia matrella, etc.), bermuda grasses(Cynodon dactylon, etc.), bent grasses (Agrostis alba, creeping bentgrass, hiland bent, etc.), blueglasses (meadow grass, bird grass, etc.),fescue (tall fescue, chewings fescue, creeping red fescue, etc.),ryegrasses (darnel, rye grass, etc.), orchard grass, timothy grass.

Others: flowers (rose, carnation, chrysanthemum, prairie gentian,gypsophila, gerbera, marigold, salvia, petunia, verbena, tulip, aster,gentian, lily, pansy, cyclamen, orchid, convallaria, lavender, stock,ornamental cabbage, primula, poinsettia, gladiolus, cattleya, daisy,cymbidium, begonia, etc.), bio-fuel plants (Jatropha, safflower,camelina, switchgrass, Miscanthus, reed canary grass, giant reed, kenaf,cassava, willow, etc.), ornamental plants.

The present invention is preferably applied to cereal crops or millets.The present invention is more preferably applied to corn, wheat,sorghum, and soybean.

In the present invention, the variety of plant is not limited so long asit is commonly cultivated. The plants of such varieties include plantswhich have been conferred with one or more useful trait by a classicalbreeding technique or a genetic engineering technique (geneticallymodified plant) as well as stack varieties obtained by crossing suchgenetically modified plants.

Such useful characters include tolerance to herbicide, pest resistance,disease resistance, stress tolerance, and improved quality of crops suchas modified fatty acid residue composition of oils and fats.

Examples of the genetically modified plant include those listed in thegenetically modified crop registration database (GM APPROVAL DATABASE)in the electronic information site (http://www.isaaa.org/) of theINTERNATIONAL SERVICE for the ACQUISITION of AGRI-BIOTECH APPLICATIONS(ISAAA). More specifically, the plant may be a plant which has beenconferred with an environmental stress tolerance, a disease resistance,a herbicide tolerance, a pest resistance or the like, or a plant whereinits trait has been modified with respect to growth and yield, quality ofproduct, sterility or the like, by genetic recombination technology.

Examples of the plant conferred with a herbicide tolerance by generecombination technology include genetically modified plants conferredwith a tolerance to protoporphyrinogen oxidase (herein after referred toas PPO) herbicides such as flumioxazin; 4-hydroxyphenyl pyruvic aciddioxygenase (hereinafter abbreviated as HPPD) inhibitors such asisoxaflutole, mesotrione; acetolactate synthase (hereinafter referred toas ALS) inhibitors such as imazethapyr, thifensulfuron methyl;5-enolpyruvylshikimate-3-phosphate synthase (hereinafter referred to asEPSP) inhibitors such as glyphosate; glutamine synthetase inhibitorssuch as glufosinate; auxin herbicides such as 2,4-D, dicamba; andherbicides such as bromoxynil.

Examples of the plant conferred with a herbicide tolerance by generecombination technology include glyhosate-tolerant genetically modifiedplants which have been introduced with one or more gene selected fromglyphosate tolerant EPS PS gene (CP4 epsps) from Agrobacteriumtumefaciens strain CP4; glyphosate metabolizing enzyme gene (gat4601,gat6421) which is a gene of glyphosate metabolizing enzyme (glyphosateN-acetyl transferase) from Bacillus (Bacillus licheniformis) modified bygene shuffling to enhance the metabolic activity; glyphosatemetabolizing enzyme (glyphosate oxidase gene, goxv247) from Ochrobactrum(Ochrobactrum anthropi strain LBAA), or EPSPS gene havingglyphosate-tolerant mutation (mepsps, 2mepsps) from corn. There areglyphosate-tolerant genetically modified varieties with respect toplants such as corn (Zea mays L.), soybean (Glycine max L.), cotton(Gossypium hirsutum L.), sugar beet (Beta vulgaris), canola (Brassicanapus, Brassica rapa), alfalfa (Medicagosativa), potato (Solanumtuberrosum L), wheat (Triticum aestivum), and creeping bent grass(Agrostis stolonifera).

Some glyphosate-tolerant genetically modified plants are commerciallyavailable. For example, a genetically modified plant expressingglyphosate-tolerant EPSPS from Agrobacterium has been marketed under thetrade name such as Roundup Ready®, a genetically modified plantexpressing glyphosate metabolizing enzyme from Bacillus with enhancedmetabolic activity by gene shuffling has been marketed under the tradename such as Optimum® GAT®, Optimum® Gly canola, and a geneticallymodified plant expressing EPSPS gene having glyphosate-tolerant mutationhas been marketed under the trade name GlyTol®.

Examples of plants conferred with herbicide-tolerance by generecombination technology include glufosinate-tolerant geneticallymodified plants which have been introduced with phosphinothricinN-acetyltransferase (PAT) gene (bar) of the glufosinate metabolizingenzyme from Streptomyces (Streptomyces hygroscopicus), phosphinothricinN-acetyltransferase gene (pat) of the glufosinate metabolizing enzymefrom Streptomyces (Streptomyces viridochromogenes), a synthesized patgene, or the like. There are glufosinate-tolerant genetically modifiedvarieties with respect to plants such as corn, soybean, cotton, canola,rice (Oryza sativa L.), sugar beet, and cichory (Cichori intybus).

Some glufosinate-tolerant genetically modified plants are commerciallyavailable. A genetically modified plant expressing glufosinatemetabolizing enzyme (bar, pat) from Streptomyces has been marked under atrade name including LibertyLink®.

Examples of herbicide-tolerant genetically modified plants includegenetically modified plants which have been introduced with the gene(bxn) of nitrilase, which is a bromoxynil-metabolizing enzyme fromKlebsiella (Klebsiella pneumoniae subsp. Ozaenae). Bromoxynil-tolerantgenetically modified varieties have been produced for plants such ascanola, cotton, tobacco (Nicotiana tabacum L.) and have been markedunder a trade name including Navigator® canola, or BXN®.

Examples of herbicide-tolerant genetically modified plants includegenetically modified carnation (Dianthus caryophyllus) which has beenintroduced with ALS herbicide-tolerant ALS gene (SurB, S4-HrA) fromtobacco as a selectable marker. Also, a genetically modified larvae(Linum usitatissumum L.) which has been introduced with ALSherbicide-tolerant ALS gene from Arabidopsis (Arabidopsis thaliana) hasbeen developed under the trade name CDC Triffid Flax. Also, agenetically modified soybean which has been introduced with ALSherbicide-tolerant ALS gene (csr1-2) from Arabidopsis has been developedunder the trade name Cultivance®. Furthermore, there aresulfonylurea/imidazolinone herbicide-tolerant genetically modified cornwhich has been introduced with ALS herbicide-tolerant ALS gene (zm-hra)from corn, and sulfonylurea herbicide-tolerant genetically modifiedsoybean which has been introduced with ALS herbicide-tolerant ALS gene(gm-hra) from soybean.

Examples of plants conferred with herbicide-tolerance by generecombination technology include isoxaflutole-tolerant geneticallymodified soybean which has been introduced with HPPD herbicide-tolerantHPPD gene (hppdPFW 336) from Pseudomonas (Pseudomonas fluorescens strainA32) and mesotrione-tolerant genetically modified soybean which has beenintroduced with HPPD gene (avhppd-03) from oats (Avena sativa).

Examples of plants conferred with herbicide-tolerance by generecombination technology include 2,4-D-tolerant genetically modifiedcorns, genetically modified soybeans, genetically modified cottons whichhave been introduced with gene (aad-1) of 2,4-D metabolizing enzymearyloxyalkanoate dioxygenase from Sphingobium (Sphingobiumherbicidovorans) or with gene (aad-12) of 2,4-D metabolizing enzymearyloxyalkanoate dioxygenase from Delftia (Delftia acidovorans). Some ofthem are developed under the trade names such as Enlist® Maize, Enlist®Soybean. Also, there are dicamba-tolerant genetically modified soybeansand cottons which have been introduced with gene (dmo) of dicambamonooxygenase, which is dicamba metabolizing enzyme fromStenotrophomonas (Stenotrophomonas maltophilia strain DI-6).

Examples of genetically modified plant tolerant to two or moreherbicides include genetically modified cotton and genetically modifiedcorn, which are tolerant to both glyphosate and glufosinate, andmarketed under the trade name such as GlyTol® LibertyLink®, RoundupReady® LibertyLink® Maize. Also, there are a genetically modifiedsoybean tolerant to both glufosinate and 2,4-D and developed under thetrade name Enlist® Soybean, and a genetically modified cotton tolerantto both glufosinate and 2,4-D. A genetically modified soybean tolerantto both glyphosate and dicamba has been developed under the trade nameGenuity®) Roundup Ready® 2 Xtend®. Genetically modified corn and soybeanresistant to both glyphosate and ALS inhibitors have been developedunder the trade name Optimum GAT®. In addition, a genetically modifiedcotton tolerant to both glufosinate and dicamba, a genetically modifiedcorn tolerant to both glyphosate and 2,4-D, a genetically modifiedsoybean tolerant to both glyphosate and HPPD herbicide have also beendeveloped. Furthermore, a genetically modified soybean tolerant to threeherbicides glyphosate, glufosinate and 2,4-D has been developed.

Examples of the plant conferred with a pest resistance by generecombination technology include plants conferred with resistance tolepidopteran insects, coccinella insects, multipter insects, nematodesand the like.

Examples of the plant conferred with a pest resistance to lepidopteraninsects by genetic recombination technology include genetically modifiedplants such as soybean, cotton, rice, poplar (Populus sp.), and tomato(Lycopersicon esculentum), and eggplant (Solanum melongena), which havebeen introduced with a gene encoding delta-endotoxin, which is aninsecticidal protein derived from a soil bacterium Bacillusthuringiensis bacteria (hereinafter referred to as Bt bacteria).Examples of the delta-endotoxin that confers a pest resistance tolepidopteran insects include Cry1A, Cry1Ab, modified Cry1Ab (truncatedCry1Ab), Cry1Ac, Cry1Ab-Ac (hybrid protein of Cry1Ab and Cry1Ac), Cry1C,Cry1F, Cry1Fa2 (modified cry1F), moCry1F (modified Cry1F), Cry1A. 105(hybrid protein of Cry1Ab, Cry1Ac and Cry1F), Cry2Ab2, Cry2Ae, Cry9C,Vip3A, Vip3Aa20, and the like.

Examples of the plant conferred with a pest resistance to coccinellainsects by genetic recombination technology include genetically modifiedplants such as corn, potato, which have been introduced with a geneencoding delta-endotoxin, which is an insecticidal protein derived froma soil bacterium Bt bacteria. Examples of the delta-endotoxin thatconfers a pest resistance to coccinella insects include Cry3A, mCry3A(modified Cry3A), Cry3Bb1, Cry34Ab1, and Cry35Ab1.

Examples of the plant conferred with a pest resistance to multipterinsects by genetic recombination technology include genetically modifiedcorn, which has been introduced with a synthetic gene encoding a hybridprotein eCry3.1Ab, which is a hybrid protein of Cry3A and Cry1Ab derivedfrom soil bacteria Bt bacteria, a genetically modified cotton, which hasbeen introduced with a gene encoding trypsin inhibitor CpTI fromblack-eyed pea (Vigna unguiculata), a genetically modified poplar, whichhas been introduced with a gene encoding API, which is a proteaseinhibitor protein A from arrowhead (Sagittaria sagittifolia).

Examples of the insecticidal protein that confers a pest resistance tothe plants include hybrid proteins, truncated proteins, and modifiedproteins of the insecticidal proteins described above. The hybridproteins are produced by combining different domains of multipleinsecticidal proteins using a common recombination technology, andCry1Ab-Ac and Cry1A.105 are known.

Examples of the truncated proteins include Cry1Ab lacking the amino acidsequence partially. Examples of the modified proteins include proteinsin which one or more amino acids of natural delta-endotoxin have beensubstituted, such as Cry1Fa2, moCry1F, mCry3A.

Examples of other insecticidal proteins that confer insect resistance toplants by genetic recombination technology include insecticidal proteinsfrom Bacillus cereus or Bacillus popilliae, the insecticidal proteinsVip 1, Vip 2, Vip 3 of Bt bacteria, insecticidal proteins from nematode,toxin produced by an animal such as scorpotoxin, spider toxin, bee venomor insect-specific neurotoxin, toxins of filamentous fungi, plantlectin, agglutinin, protease inhibitor such as trypsin inhibitor, serineprotease inhibitor, patatin, cystatin, papain inhibitor, ribosomeinactivating protein (RIP) such as ricin, corn-RIP, abrin, rufin,saporin, bryodin, steroid metabolizing enzymes such as 3-hydroxysteroidoxidase, ecdysteroid-UDP-glucosyltransferase, cholesterol oxidase,ecdysone inhibitor, HMG-CoA reductase, ion channel inhibitors such assodium channel inhibitor, calcium channel inhibitor, juvenile hormoneesterase, diuretic hormone receptor, stilbene synthase, bibenzylsynthase, chitinase, glucanase, and the like.

Genetically modified plants conferred with a pest resistance byintroducing one or more insecticidal protein gene are known, and some ofsuch genetically modified plants are commercially available.

Examples of commercially available genetically modified cotton conferredwith a pest resistance include Bollgard® cotton expressing theinsecticidal protein Cry1Ac of Bt bacteria, Bollgard II® cottonexpressing the insecticidal proteins Cry1Ac and Cry2Ab of Bt bacteria,Bollgard III® expressing the insecticidal proteins Cry1Ac, Cry2Ab, Vip3Aof Bt bacteria, VIPCOT® expressing the insecticidal proteins Vip3A andCry1Ac of Bt bacteria, WideStrike® expressing the insecticidal proteinsCry1Ac, Cry1F of Bt bacterium.

Examples of commercially available genetically modified corn conferredwith a pest resistance include YieldGard® Rootworm RW expressing theinsecticidal protein Cry3Bb1 of Bt bacteria, YieldGard Plus® expressingthe insecticidal proteins Cry1Ab and Cry3Bb1 of Bt bacteria, YieldGard®VT Pro® expressing the insecticidal proteins Cry1A. 105 and Cry2Ab2 ofBt bacteria. Agrisure® RW expressing the insecticidal protein mCry3A ofBt bacteria, Agrisure® Viptera expressing the insecticidal proteinVip3Aa20 of Bt bacteria, Agrisure® Duracade® expressing the insecticidalprotein eCry3.1Ab of Bt bacteria are also commercially available.

Examples of commercially available genetically modified potato conferredwith a pest resistance include Atlantic NewLeaf® potato, NewLeaf® RussetBurbank potato, and the like, which express the insecticidal proteinCry3A of Bt bacteria.

Examples of genetically modified plants conferred with resistance topests include kidney bean (Phaseolus vulgaris), papaya (Carica papaya),plum (Prunus domestical), potato, squash (Cucurbita pepo), sweet pepper(Capsicum annuum), tomato, and the like, which have been conferred witha resistance to plant viral diseases. Specific examples of geneticallymodified plants conferred with a resistance to plant viral diseasesinclude a genetically modified kidney bean which has been introducedwith a gene that produces double-stranded RNA of a replication proteinof bean golden mosaic virus, a genetically modified papaya which hasbeen introduced with a coat protein gene of papaya ringspot virus, agenetically modified potato which has been introduced with a coatprotein gene of potato virus Y or replication enzyme domain gene ofpotato leaf roll virus, a genetically modified squash which has beenintroduced with a coat protein gene of Cucumber mosaic virus, with acoat protein gene of Watermelon mosaic virus, or with a coat proteingene of Zucchini yellow mosaic virus, a genetically modified sweetpepper and transgenic tomato which has been introduced with a coatprotein gene of Cucumber mosaic virus, and the like.

A genetically modified potato conferred with a resistance to plant viraldiseases is commercially available under a trade name includingNewLeaf®.

Examples of the plant conferred with a resistance to pest also includeplants that have been conferred with an ability to produce a selectiveanti-pathogenic substance using genetic recombination technology. PRproteins are known as an anti-pathogenic substance (PRPs, EP392225).Such anti-pathogenic substance and genetically modified plants thatproduce the same are described in EP 392225, WO 199533818, EP 353191 andthe like. Examples of the anti-pathogenic substance include ion channelinhibitors such as sodium channel inhibitors, calcium channel inhibitors(KP1, KP4, KP6 toxin produced by viruses are known), anti-pathogenicsubstances produced by microorganisms such as stilbene synthase,bibenzyl synthase, chitinase, glucanase, peptide antibiotics,antibiotics having heterocycles, protein factors involved in pestresistance, which is referred to as pest resistance genes and describedin WO 2003000906.

Examples of genetically modified plant wherein the quality of producthas been modified includes genetically modified plants having amodification in lignin production, a modification in oils or fatty acidcomponents, production of phytic acid degrading enzymes, a modificationin flower color, a modification in alpha-amylase activity, amodification in amino acids, a modification in starch or carbohydratecomponents, inhibition of acrylamide production, reduction of blackspots due to mechanical damage, anti-allergy, reduction of nicotineproduction, or retardation of aging or grain-filling.

There is a genetically modified alfalfa wherein the lignin content hasbeen lowered by RNA interference with a gene that generatesdouble-stranded RNA of S-adenosyl-L-methionine: trans-caffeoyl CoA3-methyltransferase (ccomt) gene of alfalfa related to ligninproduction.

A genetically modified canola wherein the triacylglyceride content,including lauric acid, has been increased by introducing a gene involvedin fatty acid synthesis, 12:0 ACP thioesterase gene of laurier(Umbellularia californica), has been developed under the trade nameLaurical® Canola.

A genetically modified canola wherein the degradation of endogenousphytic acid has been enhanced by introducing a gene (phyA) of 3-phytase,which is a degrading enzyme of phytic acid of plants from Aspergillusniger, has been developed under the trade name Phytaseed® Canola. Also,a genetically modified corn wherein the degradation of endogenous phyticacid has been enhanced by introducing 3-phytase gene (phyA) ofAspergillus niger has been developed.

A genetically modified carnation wherein the flower color has beencontrolled to blue by introducing a gene of dihydroflavonol-4-reductase,which is an enzyme that produces blue pigment delphinidin and itsderivative of petunia (Petunia hybrida), and aflavonoid-3′,5′-hydroxylase gene from petunia, pansy (Violawittrockiana), salvia (Salvia splendens) or carnation is known.Genetically modified carnations with flower color controlled to bluehave been developed under the trade name such as Moonldust®,Moonshadow®, Moonshade®, Moonlite®, Moonaqua®, Moonvista®, Moonique®,Moonpearl®, Moonberry Registered trademark), and Moonvelvet®. Also,genetically modified roses with flower color controlled to blue byintroducing a gene of anthocyanin-5-acyltransferase, which is an enzymethat produces blue pigment delphinidin and its derivative, from Torenia(Torenia sp.), and a flavonoid-3′,5′-hydroxylase gene from pansy havebeen developed.

A genetically modified corn wherein the production of bioethanol hasbeen increased by introducing a gene (Amy797E) of heat-resistantalpha-amylase relating to starch degradation of Thermococcales sp. havebeen developed under the trade name Enogen®.

A genetically modified corn wherein the production of lysine has beenincreased by introducing a gene (cordapA) of dihydrodipicolinatesynthase relating to the production of amino acid lysine ofCorynebacterium glutamicum has been developed under the trade nameincluding Mavera®.

A genetically modified melon and a genetically modified tomato whereinthe shelf life has been improved by introducing a gene (sam-K) ofS-adenosylmethionine hydrolase relating to ethylene production by planthormones from Escherichia coli bacteriophage T3 has been developed.Also, genetically modified tomatoes with improved shelf life byintroducing a gene that lacks a part of the ACC synthase gene, which isinvolved in the ethylene production by plant hormones, from tomato, anACC deaminase gene from Pseudomonas (Pseudomonas chlororaphis) thatdegrades the ethylene precursor ACC, a gene that generatesdouble-stranded RNA of polygalacturonase genes which degrades cell wallpectin, or ACC oxidase genes of tomato related to the production ofethylene have been developed. A genetically modified tomato withimproved shelf life by introducing a gene that produces double-strandedRNA of polygalacturonase genes of tomato has been developed under thetrade name FLAVR SAVR®.

A genetically modified potato, wherein the possibility of decompositionof starch, formation of black spots due to mechanical damage andproduction of a carcinogen (acrylamide) from heating are lowered byintroducing a gene that generates double-stranded RNA of a transcriptionfactor promoting degradation of starch derived from potato, and a genethat generates double-stranded RNA of polyphenol oxidase gene and a genethat generates double-stranded RNA of genes involved in asparagineproduction from potato, has been developed under a trade mark includingInnate®. Also, a genetically modified potato wherein the amylose contentis lowered by introducing an antisense gene of starch synthase frompotato has been developed under the trade name Amflora®.

A genetically modified rice having alleviation effect on pollinosis withimmune tolerance by introducing a gene (7crp) of altered antigenicprotein of cedar pollen has been developed.

A genetically modified soybean wherein the oleic acid content isincreased by introducing a partial gene (gm-fad2-1) of ω-6 desaturase,which is a fatty acid desaturase enzyme, of soybean to inhibit the geneexpression thereof has been developed under the trade name Plenish® orTresis®. Also, a genetically modified soybean wherein the saturatedfatty acid content is lowered by introducing a gene (fatb1-A) thatgenerates a double-stranded RNA of acyl-acyl carrierprotein-thioesterase and a gene (fad2-1A) that generates adouble-stranded RNA of δ-12 desaturase has been developed under thetrade name Vistive Gold®. Also, a genetically modified soybean whereinthe ω3 fatty acid content is enhanced by introducing a δ-6 desaturasegene (Pj.D6D) of primrose and a δ-12 desaturase gene (Nc.Fad3) ofNeurospora crassa has been developed.

A genetically modified tobacco wherein the nicotine content is loweredby introducing an antisense gene of quinolinic acidphosphoribosyltransferase (NtQPT1) of tobacco has been developed.

A genetically modified rice, Golden rice, introduced with a phytoenesynthase gene (psy) of trumpet narcissus (Narcissus pseudonarcissus) anda carotene desaturase gene (crtl) of soil bacteria that synthesizescarotenoids (Erwinia uredovora), which allow endosperm-specificexpression to produce β-carotene in endosperm tissue, whereby a ricecontaining vitamin A is enabled to be harvested, has been developed.

Examples of the plants in which the fertile trait has been modified by agenetic recombination technique include genetically modified plantsconferred with male sterility and fertility restoration. There aregenetically modified corn and chicory conferred with male sterility byintroducing anther tapetum cell expressing a ribonuclease gene (barnase)of Bacillus (Bacillus amyloliquefaciens). There is also a geneticallymodified corn conferred with male sterility by introducing a DNA adeninemethyltransferase gene (dam) of Escherichia coli. Furthermore, there isa genetically modified corn wherein the sterility has been controlled byintroducing alpha-amylase gene (zm-aa1) of corn that confers malesterility and ms45 protein gene (ms45) of corn that confers fertilityrestoration.

There is a genetically modified canola conferred with a fertilityrestoring function by introducing anther tapetum cells expressing aribonuclease inhibitory protein gene (barstar) of Bacillus. In addition,there is a genetically modified canola wherein the sterility has beencontrolled by introducing a ribonuclease gene (barnase) of Bacillus thatconfers a male sterility and a ribonuclease inhibitory protein gene(barstar) of Bacillus that confers a fertility restoration.

Examples of the plants conferred with tolerance to environmental stressby a genetic recombination technique include genetically modified plantsconferred with tolerance to dryness. A dry tolerant corn which has beenintroduced with a cold shock protein gene (cspB) of Bacillus subtilishas been developed under the trade name Genuity® DroughtGard®. Also, drytolerant sugar cane which has been introduced with choline dehydrogenasegene (RmBetA) of alfalfa rhizobium (Rhizobium meliloti) or E. coli(Esherichia coli) has been developed.

Examples of the plants wherein a trait related to growth and yield hasbeen modified by genetic recombination technology include geneticallymodified plants having enhanced growth ability. For example, agenetically modified soybean which has been introduced with a gene ofArabidopsis encoding a transcription factor that controls circadianrhythm (bbx32) has been developed.

The plant according to the present invention can be a plant which hasbeen modified using other techniques than genetic recombinationtechnology. More specifically, it may be a plant which has beenconferred with tolerance to environmental stress, disease resistance,tolerance to herbicide, insect resistance, or the like, by classicalbreeding technique, genetic marker breeding technique, genome editingtechnique, or the like.

Examples of the plant wherein a tolerance to herbicide has beenconferred by classical breeding technique or genetic marker breedingtechnique include corn, rice, wheat, sunflower (Helianthus annuus),canola, and lentil beans (Lens culinaris), which are resistant toimidazolinone type ALS inhibiting herbicides, such as imazethapyr, andare marketed under the trade name Clearfield®. Also, there is STSsoybean, which is a soybean tolerant to sulfonylurea-based herbicide, asan example of plants which has been conferred with a resistance tosulfonyl-based ALS-inhibiting herbicides such as thifensulfuron methylby genetic marker breeding technique. Also, there is SR corn, which isresistant to sethoxydim, as an example of plants which has beenconferred with a resistance to acetyl CoA carboxylase inhibitor, such astrione oxime type herbicide, aryloxyphenoxypropionic acid typeherbicide, by genetic marker breeding technique.

Examples of the plants conferred with pest resistance by classic orgenetic marker breeding technique include a soybean having Rag 1(Resistance Aphid Gene 1) gene, which is an aphid resistant gene.Examples of the plants conferred with resistance to nematodes by theclassical breeding technique include a soybean conferred with aresistance to Cysto nematode, and a cotton conferred with a resistanceto Root Knot nematode.

Examples of the plants which has been conferred with a resistance topest by classic or genetic marker breeding technique include a cornwhich has been conferred with a resistant to anthracnose stalk rot, acorn which has been conferred with a resistant to Gray leaf spot, a cornwhich has been conferred with a resistant to Goss's wilt, a corn whichhas been conferred with a resistant to Fusarium stalk rot, a soybeanwhich has been conferred with a resistant to Asian soybean rust, apepper which has been conferred with a resistant to Phytophthora, alettuce which has been conferred with a resistant to powdery mildew, atomato which has been conferred with a resistant to Bacterial wilt, atomato which has been conferred with a resistant to Gemini virus, and alettuce which has been conferred with a resistant to downy mildew.

As an example of the plants which have been conferred with a toleranceto dryness by classic or genetic marker breeding technique, a drytolerant corn has been developed under the trade name such as AgrisureArtesian®, Optimum AQUA Max®.

As an example of the plants conferred with a tolerance to herbicide bygenomic editing technique, a canola conferred with a tolerance tosulfonylurea herbicide by rapid breed development technology wherein amutation to confer tolerance to sulfonylurea herbicide has introducedinto ALS gene via chimera oligonucleotides of DNA and RNA, has beendeveloped under the trade name SU Canola®.

The above plants include a variety which has been conferred with two ormore traits, such as tolerance to environmental stress, diseaseresistance, tolerance to herbicide, pest resistance, growth and yieldtraits, quality of product, and sterility, using a genetic recombinationtechnology as described above, such as a classic breeding technique, agenetic marker breeding, or a genome editing technique, as well as avariety which has been conferred with two or more traits from parents bycrossing the parents, which are genetically modified plants having sameor different characteristic. Examples of such plant include geneticallymodified plants conferred with both of tolerance to herbicide and pestresistance.

For example, as for a genetically modified plant conferred withtolerance to glyphosate and pest resistance, genetically modifiedcottons, such as Roundup Ready® Bollgard® cotton, Roundup Ready®Bollgard II® cotton, Roundup Ready® Flex® Bollgard II® cotton, Bollgard®III x Roundup Ready® Flex®, and VIPCOT® Roundup Ready Flex® Cotton, havebeen developed. Also, genetically modified soybeans have been developedunder the trade name, such as Agrisure® GT/RW, Roundup Ready® YieldGard®maize, Genuity® VT Double Pro®, Genuity® VT Triple Pro®, YieldGard®,YieldGard® CB+RW, YieldGard® VT® Rootworm® RR 2, YieldGard® RW+RR,YieldGard® VT Triple, or YieldGard® Plus with RR. Furthermore, agenetically modified soybean such as Intacta® Roundup Ready® 2 Pro hasbeen developed.

For example, as for genetically modified plants conferred with toleranceto glufosinate and pest resistance, genetically modified cottons havebeen developed under the trade name, such as Widestrike® Cotton,Twinlink® Cotton, and FiberMax® LibertyLink® Bollgard II®. Also,genetically modified corns have been developed under the trade name,such as Agrisure® CB/LL, Agrisure® CB/LL/RW, Agrisure® Viptera® 2100,Agrisure® Viptera® 3100, Bt Xtra Maize, NaturGard Knockout®, Herculex®RW, Herculex® CB, Herculex® XTRA, Starlink® Maize, and Liberty Link®YieldGard® Maize.

For example, as for genetically modified plants conferred with toleranceto glyphosate and glufosinate and pest resistance, genetically modifiedcottons have been developed under the trade name, such as Widestrike®Roundup Ready® Cotton, Widestrike® Roundup Ready Flex® Cotton,Widestrike® Cotton, Registered trademark) x Roundup Ready Flex® xVIPCOT® Cotton, and Glytol® x Twinlink®. Also, genetically modifiedcorns have been developed under the trade name, such as Agrisure®GT/CB/LL, Agrisure® 3000GT, Agrisure® 3122, Agrisure® Viptera® 3110,Agrisure® Viptera 3111, Agrisure® Viptera® 3220, Agrisure® Duracade®5122, Agrisure® Duracade® 5222, Optimum® Intrasect, Optimum® TRIsect,Optimum® Intrasect XTRA, Optimum® Intrasect Xtreme, Genuity® martStax®,Power Core®, Herculex® I RR, Herculex® RW Roundup Ready® 2, and HerculexXTRA® RR.

For example, as for genetically modified plants conferred with toleranceto bromoxynil and pest resistance, a genetically modified cottons hasbeen developed under the trade name, such as BXN® Plus Bollgard® Cotton.

Examples of a variety conferred with two or more traits includegenetically modified plants conferred with disease resistance and pestresistance. For example, as for genetically modified plants conferredwith resistance to potato virus Y and pest resistance, geneticallymodified potatoes have been developed under the trade name, such asHi-Lite NewLeaf® Y Potato, NewLeaf® Y Russet Burbank Potato, and ShepodyNewLeaf® Y potato. As for genetically modified plants conferred withresistance to potato leaf roll virus and pest resistance, geneticallymodified potatoes have been developed under the trade name, such asNewLeaf® Plus Russet Burbank Potato.

Examples of a variety conferred with two or more traits includegenetically modified plants conferred with tolerance to herbicide andaltered product quality. For example, a genetically modified canola andgenetically modified corn, which have been conferred with tolerance toglufosinate and fertile trait have been developed under the trade name,such as InVigor® Canola and InVigor® Maize, respectively.

Examples of a variety conferred with two or more traits includegenetically modified plants conferred with a pest resistance and alteredproduct quality. For example, a genetically modified corn conferred withresistance to lepidopterous insects and a trait of enhanced lysineproduction has been developed under the trade name such as Mavera®YieldGard® Maize.

For other Examples of a variety conferred with two or more traits asmentioned above, genetically modified plants conferred with tolerance toherbicide and a trait altering fertility, genetically modified plantsconferred with tolerance to herbicide and tolerance to environmentalstress, genetically modified plants conferred with tolerance toherbicide and a trait modifying growth and yield, genetically modifiedplants conferred with tolerance to herbicide, pest resistance, and atrait modifying product quality, genetically modified plants conferredwith tolerance to herbicide, pest resistance, and tolerance toenvironmental stress, have been developed.

Examples of the pest which can be controlled according to the presentinvention include the followings.

Hemipteran pests: planthoppers such as small brown planthopper(Laodelphax striatellus); leafhoppers such as Empoasca onukii); aphidssuch as cotton aphid (Aphis gossypil), green peach aphid (Myzuspersicae), cabbage aphid (Brevicoryne brassicae), potato aphid(Macrosiphum euphorbiae), greenhouse potato aphid (Aulacorthum solani),bird-cherry oat aphid (Rhopalosiphum padi), pea aphid (Acyrthosiphonpisum), water lily aphid (Rhopalosiphum nymphaeae), Aphis naturtii, andAphis fabae; pentatomids such as southern green stink bug (Nezaraviridula), brown-marmorated stink bug (Halyomorpha mista) and tarnishedplant bug (Lygus lineolaris); and whiteflies such as greenhouse whitefly(Trialeurodes vaporariorum), sweetpotato whitefly (Bemisia tabaci), andsilverleaf whitey (Bemisia argentifolii).

Lepidopteran pests: pyralids such as Asian corn borer (Ostriniafurnacalis), cabbage webworm (Hellula undalis), bluegrass webworm(Pediasia teterrellus), and European corn borer (Ostrinia nubilaris);noctuids such as common cutworm (Spodoptera litura), black cutworm(Agrotis ipsilon), and oriental armyworm (Mythimna separata); pieridbutterflies such as small white butterfly (Pieris rapae); tortricidssuch as sugarcane shoot borer (Tetramoera schistaceana); ermine mothssuch as diamondback moth (Plutella xylostella); and gelechiids such aspotato tuber moth (Phthorimaea operculella).

Thysanopteran pests: thrips such as western flower thrips (Frankliniellaoccidentalis), southern yellow thrips (Thrips parmi), yellow tea thrips(Scirtothrips dorsalis), onion thrips (Thrips tabaci), flower thrips(Frankliniella intonsa), and tobacco thrips (Frankliniella fusca).

Dipteran pests: anthomylid files such as bean seed fly (Delia platura)and onion fly (Delia antiqua); leafminer flies such as vegetableleafminer (Liriomyza sativae), American serpentine leafminer (Liriomyzatrifolii), and garden pea leafminer (Chromatomyia horticola).

Coleopteran pests: corn rootworms (Diabrotica spp.) such as western cornrootworm (Diabrotica virgifera virgifera) and southern corn rootworm(Diabrotica undecimpunctata howardi); scarab beetles such as Cupreuschafer (Anomala cuprea), green chafer beetle (Anomala albopilosa),soybean beetle (Anomala rufocuprea), and Japanese beetle (Popilliajaponica); weevils such as brown gourd-shaped weevil (Sphenophorusuniformis); leaf beetles such as cucurbit leaf beetle (Aulacophorafemoralis), Colorado potato beetle (Leptinotarsa decemlineata) and beanleaf beetle (Cerotoma trifurcate); and click beetles (Agriotes spp.).

The present invention is preferably applicable to Hemipteran pests,Lepidopteran pests, Dipteran pests, and Coleopteran pests, and isparticularly preferably applicable to click beetles (Agriotes spp.),corn rootworms (Diabrotica spp.), noctuids, anthomylid flies, andaphids.

EXAMPLES

The invention is described in more detail with reference to thefollowing Preparation Examples, Formulation Examples, Seed TreatmentExamples, and Test Examples, which are not intended to limit the scopeof the present invention. The term “part” means “part by weight” unlessotherwise specified.

Preparation Examples are provided below.

Preparation Example 1

A culture broth of the present bacterial strain, which has been culturedby a known technique, is centrifuged according to an ordinary method toseparate into a supernatant and a precipitate. The supernatant isremoved, and the precipitate is washed with sterilized water to obtain abacterial mass. The obtained bacterial mass is suspended in water, driedon spray drier, and the resultant dried product is pulverized to obtaina powder of the present bacterial strain.

Preparation Example 2

A culture broth of the present bacterial strain, which has been culturedby a known technique, is frozen at −80° C., freeze-dried and pulverizedto obtain a powder of the present bacterial strain.

Preparation Example 3

In a 500 mL Erlenmeyer flask with baffle, a platinum loop scraping ofthe present bacterial strain, which have been cultured in TSA (an agarmedium containing 15 g/L of casein peptone, 5 g/L of soybean peptone, 5g/L of sodium chloride, and 15 g/L of agar), are inoculated to a liquidmedium containing 200 mL TSB (a liquid medium containing 17 g/L, ofcasein peptone, 3 g/L of soybean peptone, 2.5 g/L of glucose, 5 g/L ofsodium chloride and 2.5 g/L of K₂HPO₄) and incubated at 30° C. for 12hours to 24 hours to obtain a liquid culture. Ina 500 mL volumeErlenmeyer flask with baffle, 2 mL of the liquid culture is inoculatedto 200 mL of a fresh TSB and cultured with shaking for 24 hours to 48hours to obtain a liquid culture of the present bacterial strain(hereinafter referred to as Liquid Culture a). The Liquid Culture a iscentrifuged according to a conventional manner to separate into asupernatant and precipitate. After removing the supernatant, theprecipitate is washed with sterile water and centrifuged. Thesupernatant is removed to obtain bacterial cells of the presentbacterial strain.

Preparation Example 4

The bacterial cells of the present bacterial strain obtained inPreparation Example 3 are suspended in water, dried on spray drier, andpulverized the resulting dried product to obtain a powder of the presentbacterial strain.

Preparation Example 5

The Liquid Culture a is obtained as described in Preparation 3. TheLiquid Culture a is frozen at −80° C., and freeze-dried and pulverizedto obtain a powder of the present bacterial strain.

Preparation Example 6

In a Erlenmeyer flask with baffle, a platinum loop scraping of thepresent bacterial strain, which have been cultured in TSA (an agarmedium containing 15 g/L of casein peptone, 5 g/L of soybean peptone, 5g/L of sodium chloride, and 15 g/L of agar), were inoculated to a liquidmedium containing 200 mL TSB (a liquid medium containing 17 g/L ofcasein peptone, 3 g/L of soybean peptone, 2.5 g/L of glucose, 5 g/L ofsodium chloride and 2.5 g/L of K₂HPO₄) and incubated at 30° C. for 23hours to obtain a liquid culture. The liquid culture (2% (v/v)) wasinoculated to a fresh TSB in a Erlenmeyer flask with baffle and culturedat 30° C. with shaking for 43 hours to obtain a liquid culture of thepresent bacterial strain (hereinafter referred to as Liquid Culture b).The liquid culture b was centrifuged at 1900×g for 10 min to separateinto a supernatant and a precipitate. After removing the supernatant,the precipitate was washed with sterilized water and centrifuged at1900×g for 10 min. The supernatant was removed to obtain 3.8×10¹¹ cfu/gof bacterial cells of the present bacterial strain.

Preparation Example 7

The bacterial cells of the present bacterial strain obtained asdescribed in Preparation Example 6 were frozen at ˜80° C. andfreeze-dried. The dried product obtained thus by freeze-drying waspulverized using scoopula to obtain 2.8×10¹² cfu/g of a powder of thepresent bacterial strain.

Formulation Examples are provided below.

Formulation Example 1

To a mixture containing 4 parts of clothianidin, 5 parts of whitecarbon, 8 parts of sodium lignin sulfonate, 2 parts of sodium alkylnaphthalene sulfonate are added the powder of the present bacterialstrain obtained as described in Preparation Example 1 or 2, in theamount of 1×10¹⁰ cfu per 1 g of the formulation, and diatomaceous earthto 100 parts, followed by mixing and grinding to obtain wettable powder.

Formulation Example 2

To a mixture containing 4.5 parts of flupyradifurone, 5 parts of whitecarbon, 8 parts of sodium lignin sulfonate, 2 parts of sodium alkylnaphthalene sulfonate are added the powder of the present bacterialstrain obtained as described in Preparation Example 1 or 2, in theamount of 1×10¹⁰ cfu per 1 g of the formulation, and diatomaceous earthto 100 parts, followed by mixing and grinding to obtain wettable powder.

Formulation Example 3

To a mixture containing 7.5 parts of imidacloprid, 5 parts of whitecarbon, 8 parts of sodium lignin sulfonate, 2 parts of sodium alkylnaphthalene sulfonate are added the powder of the present bacterialstrain obtained as described in Preparation Example 1 or 2, in theamount of 1×10¹⁰ cfu per 1 g of the formulation, and diatomaceous earthto 100 parts, followed by mixing and grinding to obtain wettable powder.

Formulation Example 4

To a mixture containing 5 parts of thiacloprid, 5 parts of white carbon,8 parts of sodium lignin sulfonate, 2 parts of sodium alkyl naphthalenesulfonate are added the powder of the present bacterial strain obtainedas described in Preparation Example 1 or 2, in the amount of 1×10¹⁰ cfuper 1 g of the formulation, and diatomaceous earth to 100 parts,followed by mixing and grinding to obtain wettable powder.

Formulation Example 5

To a mixture containing 4 parts of thiamethoxam and 5 parts of whitecarbon, 8 parts of sodium lignin sulfonate, 2 parts of sodium alkylnaphthalene sulfonate are added the powder of the present bacterialstrain obtained as described in Preparation Example 1 or 2, in theamount of 1×10¹⁰ cfu per 1 g of the formulation, and diatomaceous earthto 100 parts, followed by mixing and grinding to obtain wettable powder.

Formulation Example 6

To a mixture containing 20 parts of clothianidin and 30 parts of whitecarbon containing 30% by weight of polyoxyethylene alkyl ether sulfateammonium salt are added the powder of the present bacterial strainobtained as described in Preparation Example 1 or 2, in the amount of1×10¹⁰ cfu per 1 g of the formulation, and water to 100 parts, followedby wet-milling to finely milled to obtain a flowable formulation.

Formulation Example 7

To a mixture containing 22.5 parts of flupyradifurone and parts of whitecarbon containing 30% by weight of polyoxyethylene alkyl ether sulfateammonium salt are added the powder of the present bacterial strainobtained as described in Preparation Example 1 or 2, in the amount of1×10¹⁰ cfu per 1 g of the formulation, and water to 100 parts, followedby wet-milling to finely milled to obtain a flowable formulation.

Formulation Example 8

To a mixture containing 37.5 parts of imidacloprid and parts of whitecarbon containing 30% by weight of polyoxyethylene alkyl ether sulfateammonium salt are added the powder of the present bacterial strainobtained as described in Preparation Example 1 or 2, in the amount of1×10¹⁰ cfu per 1 g of the formulation, and water to 100 parts, followedby wet-milling to finely milled to obtain a flowable formulation.

Formulation Example 9

To a mixture containing 25 parts of thiacloprid and 30 parts of whitecarbon containing 30% by weight of polyoxyethylene alkyl ether sulfateammonium salt are added the powder of the present bacterial strainobtained as described in Preparation Example 1 or 2, in the amount of1×10¹⁰ cfu per 1 g of the formulation, and water to 100 parts are addedand finely milled by wet-milling to obtain a flowable formulation.

Formulation Example 10

To a mixture containing 20 parts of thiamethoxam and 30 parts of whitecarbon containing 30% by weight of polyoxyethylene alkyl ether sulfateammonium salt are added the powder of the present bacterial strainobtained as described in Preparation Example 1 or 2, in the amount of1×10¹⁰ cfu per 1 g of the formulation, and water to 100 parts, followedby wet-milling to finely milled to obtain a flowable formulation.

Formulation Example 11

To a mixture containing 5 parts of white carbon, 8 parts of sodiumlignin sulfonate, and 2 parts of sodium alkyl naphthalene sulfonate areadded the bacterial cells or powder of the present bacterial strainobtained as described in any one of Preparation Examples 3 to 5, in theamount of 1×10¹⁰ cfu per 1 g of the formulation, and diatomaceous earthto 100 parts to obtain a mixture. The mixture iss milled to obtainwettable powder.

Formulation Example 12

To 30 parts of white carbon containing 30% by weight of polyoxyethylenealkyl ether sulfate ammonium salt are added the bacterial cells orpowder of the present bacterial strain obtained as described in any oneof Preparation Examples 3 to 5, in the amount of 1×10¹⁰ cfu or 1×10¹²cfu per 1 g of the formulation, and water to 100 parts to obtain amixture. The mixture is finely milled by wet-milling to obtain aflowable formulation.

Formulation Example 13

To a mixture containing 5 parts of white carbon, 8 parts of sodiumlignin sulfonate, and 2 parts of sodium alkyl naphthalene sulfonate wereadded the bacterial cells or powder of the present bacterial strainobtained as described in any one of Preparation Example 7, in the amountof 1×10¹⁰ cfu per 1 g of the formulation, and diatomaceous earth to 100parts to obtain a mixture. The mixture was finely milled to obtainwettable powder.

Formulation Example 14

To 30 parts of white carbon containing 30% by weight of polyoxyethylenealkyl ether sulfate ammonium salt were added the powder of the presentbacterial strain obtained in Preparation Example 7, in the amount of1×10¹⁰ cfu or 1×10¹² cfu per 1 g of the formulation, and water to 100parts to obtain a mixture. The mixture was finely milled by wet-millingto obtain a flowable formulation.

Seed Treatment Examples are provided below.

Seed Treatment Example 1

To a mixture containing 5 parts of white carbon, 8 parts of sodiumlignin sulfonate and 2 parts of sodium alkyl naphthalene sulfonate areadded the powder of the present bacterial strain obtained as describedin Preparation Example 1, in the amount of 1×10¹⁰ cfu per 1 g of theobtained formulation, and diatomaceous earth to 100 parts, followed bymixing and grinding to obtain wettable powder of the present bacterialstrain.

Soybean seeds are treated by smearing treatment with a mixed formulationof wettable powder of the present bacterial strain (adjusted to 1×10¹⁰cfu of the present bacterial strain per 1 kg of the soybean seeds) andimidacloprid wettable powder (10% wettable powder, trade name: ADMIREwettable powder, Bayer CropScience, adjusted to 0.75 g of imidaclopridper 1 kg of the soybean seeds).

Seed Treatment Example 2

Corn seeds are treated by smearing treatment with thiacloprid flowableformulation (40.0% flowable formulation, trade name: Sonido, BayerCropScience) in the amount of 1.7 g of thiacloprid per 1 kg of theseeds. The corn seeds thus treated with thiacloprid are treated by wetpowder coating with wettable powder of the present bacterial strainprepared as described in Seed Treatment Example 1 (adjusted to 1×10¹⁰cfu/kg of the present bacterial strain per 1 kg of said corn seeds).

Seed Treatment Example 3

To 30 parts of white carbon containing 30 parts of polyoxyethylene alkylether sulfate ammonium salt are added the powder of the presentbacterial strain obtained as described in Preparation Example 2, in theamount of 1×10¹⁰ cfu per 1 g of the formulation, and water to 100 parts,and the mixture is finely milled by wet-milling to obtain a flowableformulation.

Wheat seeds are treated by smearing treatment with a liquid mixturecontaining the flowable formulation of the present bacterial strain(1×10¹⁰ cfu of the present bacterial strain per 1 kg of the seeds) andclothianidin flowable formulation (47.8% flowable formulation, tradename: NipsIt Inside Insecticide, Valent U.S.A. Corporation, adjusted to0.6 g of clothianidin per 1 kg of the seeds).

Seed Treatment Example 4

Sorghum seeds are treated by smearing treatment with flowableformulation of the present bacterial strain prepared as described inSeed Treatment Example 3 (adjusted to 1×10¹⁰ cfu of the presentbacterial strain per 1 kg of the seeds). The Sorghum seeds thus treatedwith the present bacterial strain are treated by smearing treatment witha liquid containing thiamethoxam flowable formulation (47.6% flowableformulation, trade name: Cruiser 5FS, Syngenta Crop Protection LCC) inthe amount of 2.4 g of thiamethoxam per 1 kg of said sorghum seeds.

Seed Treatment Example 5

Soybean seeds are treated by smearing treatment with the flowableformulation of the present bacterial strain and flupyradifurone preparedas described in Formulation Example 7, in the amount of 2×10¹⁰ cfu ofthe present bacterial strain and 0.45 g of flupyradifurone per 1 kg ofthe soybean seeds.

Seed Treatment Example 6

Soybean seeds are treated by wet coating with a mixture obtained bymixing a wettable powder of the present bacterial strain obtained inFormulation Example 11 (adjusted to 1×10¹⁰ cfu of the present bacterialstrain per 1 kg of the seeds) and imidacloprid wettable powder (10%wettable powder, trade name: ADMIRE wettable powder, Bayer CropScience,adjusted to 0.75 g of imidacloprid per 1 kg of the seeds).

Seed Treatment Example 7

Wheat seeds are treated by smearing treatment with a liquid mixtureprepared by mixing a flowable formulation of the present bacterialstrain obtained as described in Formulation Example 12 and clothianidinflowable formulation (47.8% flowable formulation, trade name: NipsItInside Insecticide, Valent U.S.A. Corporation), in the amount of 1×10¹⁰cfu of the present bacterial strain and 0.6 g of clothianidin per 1 kgof the seeds.

Test Examples are provided below.

Test Example 1

A plastic pot is filled with a soil, and then, soybean seeds obtained inSeed Treatment Example 1 are seeded, and cultivation is carried out in aglass greenhouse for given days (“treated compartment”). Twenty Aphisgossypii are released on the 14th day after the seeding, and the numberof living Aphis gossypii is counted on the 5th day after the release.Based on the numbers of survivors in the treated compartment and theuntreated compartment, the effect on the treated compartment iscalculated by the following equation.Controlling effect=100×[(number of survivors in untreatedcompartment−number of survivors in treated compartment)/number ofsurvivors in untreated compartment]

The composition of the invention shows a significantly highercontrolling effect.

Test Example 2

In a rotary seed treatment machine (trade name: HEGE11, manufactured byWINTERSTEIGER), soybean seeds (variety:“Hatayutaka”) are treated bysmearing treatment with a liquid prepared by mixing flowable formulationof the present bacterial strain as obtained in Seed Treatment Example 3(adjusted to 1×10¹⁰ cfu of the present bacterial strain per 1 kg of theseeds) and clothianidin flowable formulation (47.8% flowableformulation, trade name: NipsIt Inside Insecticide, Valent U.S.A.Corporation, adjusted to 0.4 g of clothianidin per 1 kg of the seeds),or with a liquid prepared by mixing flowable formulation of the presentbacterial strain as obtained in Seed Treatment Example 3 (adjusted to1×10¹⁰ cfu of the present bacterial strain per 1 kg of the seeds) and achemical liquid prepared by dissolving flupyradifurone flowableformulation in acetone/Tween 20 (weight ratio=95:5) and diluting withwater (adjusted to 0.45 g of flupyradifurone per 1 kg of the seeds), orwith a liquid prepared by mixing flowable formulation of the presentbacterial strain as obtained in Seed Treatment Example 3 (adjusted to1×10¹⁰ cfu of the present bacterial strain per 1 kg of the seeds) andimidacloprid flowable formulation (48.7% flowable formulation, tradename: Gaucho 600, Bayer CropScience, adjusted to 0.75 g of imidaclopridper 1 kg of the seeds), or with a liquid prepared by mixing flowableformulation of the present bacterial strain as obtained in SeedTreatment Example 3 (adjusted to 1×10¹⁰ cfu of the present bacterialstrain per 1 kg of the seeds) and thiacloprid flowable formulation(40.0% flowable formulation, trade name: Sonido, Bayer CropScience,adjusted to 0.5 g of thiacloprid per 1 kg of the seeds), or with aliquid prepared by mixing flowable formulation of the present bacterialstrain as obtained in Seed Treatment Example 3 (adjusted to 1×10¹⁰ cfuof the present bacterial strain per 1 kg of the seeds) and thiamethoxamflowable formulation (47.6% flowable formulation, trade name: Cruiser5FS, Syngenta Crop Protection LCC, adjusted to 0.4 g of thiamethoxam per1 kg of the seeds).

A plastic pot is filled with a soil, and then, the seeds thus treatedwith the test compound are seeded, and cultivation is carried out in aglass greenhouse for given days (“treated compartment”). Twenty Aphisgossypii are released on the 14th day after the seeding, and the numberof living Aphis gossypii is counted on the 5th day after the release.Based on the numbers of survivors in the treated compartment and theuntreated compartment, the effect in the treated compartment iscalculated by the following equation.Controlling effect=100×[(number of survivors in untreatedcompartment−number of survivors in treated compartment)/number ofsurvivors in untreated compartment]

The composition of the invention shows a significantly highercontrolling effect.

Test Example 3

Soybean seeds (variety:“Hatayutaka”) are treated by wet powder coatingtreatment with the wettable powder of the present bacterial strain andclothianidin prepared in Formulation Example 1 (adjusted to 1×10¹¹ cfuof the present bacterial strain and 0.4 g of clothianidin per 1 kg ofthe seeds), or with the wettable powder of the present bacterial strainand flupyradifurone prepared in Formulation Example 2 (adjusted to1×10¹¹ cfu of the present bacterial strain and 0.45 g of flupyradifuroneper 1 kg of the seeds), or with the wettable powder of the presentbacterial strain and imidacloprid prepared in Formulation Example 3(adjusted to 1×10¹¹ cfu of the present bacterial strain and 0.75 g ofimidacloprid per 1 kg of the seeds), or with the wettable powder of thepresent bacterial strain and thiacloprid prepared in Formulation Example4 (adjusted to 1×10¹¹ cfu of the present bacterial strain and 0.5 g ofthiacloprid per 1 kg of the seeds), or with the wettable powder of thepresent bacterial strain and thiamethoxam prepared in FormulationExample 5 (adjusted to 1×10¹¹ cfu of the present bacterial strain and0.4 g of thiamethoxam per 1 kg of the seeds).

A plastic pot is filled with a soil, and then, the seeds thus treatedwith the test compound are seeded, and cultivation is carried out in aglass greenhouse for given days (“treated compartment”). Twenty Aphisgossypii are released on the 14th day after the seeding, and the numberof living Aphis gossypii is counted on the 5th day after the release.Based on the numbers of survivors in the treated compartment and theuntreated compartment, the effect in the treated compartment iscalculated by the following equation.Controlling effect=100×[(number of survivors in untreatedcompartment−number of survivors in treated compartment)/number ofsurvivors in untreated compartment]

The composition of the invention shows a significantly highercontrolling effect.

Test Example 4

In a rotary seed treatment machine (trade name: HEGE11, manufactured byWINTERSTEIGER), soybean seeds (variety:“Hatayutaka”) are treated bysmearing treatment with the flowable formulation of the presentbacterial strain and clothianidin prepared in Formulation Example 6(adjusted to 2×10¹⁰ cfu of the present bacterial strain and 0.4 g ofclothianidin per 1 kg of the seeds), or with the flowable formulation ofthe present bacterial strain and flupyradifurone prepared in FormulationExample 7 (adjusted to 2×10¹⁰ cfu of the present bacterial strain and0.45 g of flupyradifurone per 1 kg of the seeds) or with the flowableformulation of the present bacterial strain and imidacloprid prepared inFormulation Example 8 (adjusted to 2×10¹⁰ cfu of the present bacterialstrain and 0.75 g of imidacloprid per 1 kg of the seeds) or with theflowable formulation of the present bacterial strain and thiaclopridprepared in Formulation Example 9 (adjusted to 2×10¹⁰ cfu of the presentbacterial strain and 0.5 g of thiacloprid per 1 kg of the seeds) or withthe flowable formulation of the present bacterial strain andthiamethoxam prepared in Formulation Example 10 (adjusted to 2×10¹⁰ cfuof the present bacterial strain and 0.4 g of thiamethoxam per 1 kg ofthe seeds).

A plastic pot is filled with a soil, and then, the seeds thus treatedwith the test compound are seeded, and cultivation is carried out in aglass greenhouse for given days (“treated compartment”). Twenty Aphisgossypii are released on the 14th day after the seeding, and the numberof living Aphis gossypii is counted on the 5th day after the release.Based on the numbers of survivors in the treated compartment and theuntreated compartment, the effect in the treated compartment iscalculated by the following equation.Controlling effect=100×[(number of survivors in untreatedcompartment−number of survivors in treated compartment)/number ofsurvivors in untreated compartment]

The composition of the invention shows a significantly highercontrolling effect.

Test Example 5

In a rotary seed treatment machine (trade name: HEGE11, manufactured byWINTERSTEIGER), corn seeds (variety: yellow dent corn) are treated bysmearing treatment with a liquid containing clothianidin flowableformulation (47.8% flowable formulation, trade name: NipsIt InsideInsecticide, Valent U.S.A. Corporation, adjusted to 1.5 g ofclothianidin per 1 kg of the seeds), or with a liquid prepared bydissolving flupyradifurone in acetone/Tween 20 (weight ratio=95:5) anddiluting with water (adjusted to 1.7 g of flupyradifurone per 1 kg ofthe seeds), or with a liquid containing imidacloprid flowableformulation (48.7% flowable formulation, trade name: Gaucho 600, BayerCropScience, adjusted to 2.8 g of imidacloprid per 1 kg of the seeds),or with a liquid containing thiacloprid flowable formulation (40.0%flowable formulation, trade name: Sonido, Bayer CropScience, adjusted to3.0 g of thiacloprid per 1 kg of the seeds), or with a liquid containingthiamethoxam flowable formulation (47.6% flowable formulation, tradename: Cruiser 5FS, Syngenta Crop Protection LCC, adjusted to 1.5 g ofthiamethoxam per 1 kg of the seeds). In a rotary seed treatment machine(trade name: HEGE11, manufactured by WINTERSTEIGER), the corn seeds thustreated with clothianidin, or with flupyradifurone, or withimidacloprid, or with thiacloprid, or with thiamethoxam are treated bywet powder coating with the wettable powder of the present bacterialstrain obtained in Seed Treatment Example 1 (adjusted to 1×10¹⁰ cfu ofthe present bacterial strain per 1 kg of the seeds).

A plastic pot is filled with a soil, and then, the seeds thus treatedwith the test compound are seeded, and cultivation is carried out in aglass greenhouse for given days (“treated compartment”). Newly hatchedlarvae of Diabrotica virgifera virgifera are released (10 for each stalkof corn) on the 7th day after the seeding, and the number of livingDiabrotica virgifera virgifera is counted on the 10th day after therelease. Based on the numbers of survivors in the treated compartmentand the untreated compartment, the effect in the treated compartment iscalculated by the following equation.Controlling effect=100×[(number of survivors in untreatedcompartment−number of survivors in treated compartment)/number ofsurvivors in untreated compartment]

The composition of the invention shows a significantly highercontrolling effect.

Test Example 6

In a rotary seed treatment machine (trade name: HEGE11, manufactured byWINTERSTEIGER), corn seeds (variety: yellow dent corn) are treated bysmearing treatment with flowable formulation of the present bacterialstrain as obtained in Seed Treatment Example 3 (adjusted to 1×10¹⁰ cfuof the present bacterial strain per 1 kg of the seeds). In a rotary seedtreatment machine (trade name: HEGE11, manufactured by WINTERSTEIGER),the corn seeds thus treated with the present bacterial strain aretreated by smearing treatment with a liquid containing clothianidinflowable formulation (47.8% flowable formulation, trade name: NipsItInside Insecticide, Valent U.S.A. Corporation, adjusted to 1.5 g ofclothianidin per 1 kg of the seeds), or with a liquid prepared bydissolving flupyradifurone in acetone/Tween 20 (weight ratio=95:5) anddiluting with water (adjusted to 1.7 g of flupyradifurone per 1 kg ofthe seeds), or with a liquid containing imidacloprid flowableformulation (48.7% flowable formulation, trade name: Gaucho 600, BayerCropScience, adjusted to 2.8 g of imidacloprid per 1 kg of the seeds),or with a liquid containing thiacloprid flowable formulation (40.0%flowable formulation, trade name: Sonido, Bayer CropScience, adjusted to3.0 g of thiacloprid per 1 kg of the seeds), or with a liquid containingthiamethoxam flowable formulation (47.6% flowable formulation, tradename: Cruiser 5FS, Syngenta Crop Protection LCC, adjusted to 1.5 g ofthiamethoxam per 1 kg of the seeds).

A plastic pot is filled with a soil, and then, the seeds thus treatedwith the test compound are seeded, and cultivation is carried out in aglass greenhouse for given days (“treated compartment”). Newly hatchedlarvae of Diabrotica virgifera virgifera are released (10 for each stalkof corn) on the 7th day after the seeding, and the number of livingDiabrotica virgifera virgifera is counted on the 10th day after therelease. Based on the numbers of survivors in the treated compartmentand the untreated compartment, the effect in the treated compartment iscalculated by the following equation.Controlling effect=100×[(number of survivors in untreatedcompartment−number of survivors in treated compartment)/number ofsurvivors in untreated compartment]

The composition of the invention shows a significantly highercontrolling effect.

Test Example 7

A chemical liquid is prepared by diluting clothianidin wettable powder(16% wettable powder, trade name: DANTOTSU, Sumitomo Chemical Company,Limited) to 160 ppm of clothianidin, or by dissolving flupyradifurone inacetone/Tween 20 (weight ratio=95:5) and diluting with water to 200 ppmof flupyradifurone, or diluting wettable imidacloprid granules (50%wettable granules, trade name: Admire wettable granules, BayerCropScience) to 200 ppm of imidacloprid, or diluting wettablethiacloprid granules (30% wettable granules, trade name: BARIARDwettable granules, Bayer CropScience) to 300 ppm of thiacloprid, ordiluting thiamethoxam water soluble powder (10.0% soluble powder, tradename: Actara Water Soluble Powder, Syngenta Japan) to 66.7 ppm ofthiamethoxam, independently, and followed by combining each chemicalliquid with the equal volume of a solution of the wettable powder of thepresent bacterial strain obtained in Seed Treatment Example 1 (adjustedto 2×10⁸ cfu of the present bacterial strain per 1 L of spray liquid).

The chemical liquid is sprayed in a sufficient amount to pot plantingcucumber plants wherein primary leaf development has occurred. After airdrying, 20 Aphis gossypii are released, and the number of living Aphisgossypii is counted on the 5th day after the release. Based on thenumbers of survivors in the treated compartment and the untreatedcompartment, the effect in the treated compartment is calculated by thefollowing equation.Controlling effect=100×[(number of survivors in untreatedcompartment−number of survivors in treated compartment)/number ofsurvivors in untreated compartment]

The composition of the invention shows a significantly highercontrolling effect.

Test Example 8

Soybean seeds are treated with a wettable powder of the presentbacterial strain as obtained in Formulation Example 11 (adjusted to1×10⁸ cfu or 1×10¹⁰ cfu of the present bacterial strain per 1 kg of theseeds) to obtain soybean seeds treated with the present bacterialstrain. Also, soybean seeds are treated with wettable imidaclopridpowder (10% wettable powder, trade name: Admire wettable powder, BayerCropScience, adjusted to 0.75 g of imidacloprid per 1 kg of the seeds)to obtain soybean seed treated with imidacloprid.

A plastic pot is filled with a soil, and then, soybean seeds, which havebeen treated with the present bacterial strain and imidacloprid obtainedin Seed Treatment Example 6, or soybean seeds treated with the presentbacterial strain or with imidacloprid are seeded, and cultivation iscarried out in a glass greenhouse for given days (“treatedcompartment”). Twenty Aphis gossypii are released on the 14th day afterthe seeding, and the number of living Aphis gossypii is counted on the5th day after the release. Similar procedures are conducted usinguntreated soybean seeds, instead of the treated soybean seeds, asdescribed above for the treated compartment (“untreated compartment”).

Based on the numbers of survivors in the treated compartment and theuntreated compartment, the effect in the treated compartment iscalculated by the following equation.Controlling effect=100×[(number of survivors in untreatedcompartment−number of survivors in treated compartment)/number ofsurvivors in untreated compartment]

TABLE 1 Bacteria/Compound retained by Retaining Amount Seeds (/Kg seeds)Imidacloprid 0.75 g The present bacterial strain 1 × 10⁸ cfu The presentbacterial strain 1 × 10¹⁰ cfu The present bacterial strain 1 × 10⁸ cfuImidacloprid 0.75 g The present bacterial strain 1 × 10¹⁰ cfuImidacloprid 0.75 g

The compartment treated with the composition of the invention shows asynergistic controlling effect, compared with that of the correspondingcompartment treated solely with imidacloprid or the present bacterialstrain.

Test Example 9

In a rotary seed treatment machine (trade name: HEGE11, manufactured byWINTERSTEIGER), soybean seeds (variety:“Hatayutaka”) are treated bysmearing treatment using a flowable formulation of the present bacterialstrain as obtained in Formulation 12, and flupyradifurone flowableformulation (20.0% liquid formulation, trade name: SIVANTO200, BayerCropScience), or thiacloprid flowable formulation (40.0% flowableformulation, trade name: Sonido, Bayer CropScience), or thiamethoxamflowable formulation (47.6% flowable formulation, trade name: Cruiser5FS, Syngenta Crop Protection LCC, so that the soybean seeds retain thepresent bacterial strain and the compound in the amount shown in Table2.

A plastic pot is filled with a soil, and then, the soybean seeds, whichhave been treated with the present bacterial strain, compound or thepresent bacterial strain+compound as shown in Table 2, are seeded, andcultivation is carried out in a glass greenhouse for given days(“treated compartment”). Twenty Aphis gossypii are released on the 14thday after the seeding, and the number of living Aphis gossypii iscounted on the 5th day after the release. Similar procedures areconducted using untreated soybean seeds, instead of the treated soybeanseeds, as described above for the treated compartment (“untreatedcompartment”).

Based on the numbers of survivors in the treated compartment and theuntreated compartment, the effect in the treated compartment iscalculated by the following equation.Controlling effect=100×[(number of survivors in untreatedcompartment−number of survivors in treated compartment)/number ofsurvivors in untreated compartment]

TABLE 2 Bacteria/Compound retained Retaining Amount (/Kg by Seeds seeds)The present bacterial strain 1 × 10¹⁰ cfu The present bacterial strain 1× 10¹³ cfu Flupyradifurone  0.2 g Flupyradifurone  0.4 g Thiacloprid 0.3 g Thiacloprid 0.45 g Thiamethoxam 0.15 g Thiamethoxam 0.25 g Thepresent bacterial strain 1 × 10¹⁰ cfu Flupyradifurone  0.2 g The presentbacterial strain 1 × 10¹³ cfu Flupyradifurone  0.2 g The presentbacterial strain 1 × 10¹⁰ cfu Flupyradifurone  0.4 g The presentbacterial strain 1 × 10¹³ cfu Flupyradifurone  0.4 g The presentbacterial strain 1 × 10¹⁰ cfu Thiacloprid  0.3 g The present bacterialstrain 1 × 10¹³ cfu Thiacloprid  0.3 g The present bacterial strain 1 ×10¹⁰ cfu Thiacloprid 0.45 g The present bacterial strain 1 × 10¹³ cfuThiacloprid 0.45 g The present bacterial strain 1 × 10¹⁰ cfuThiamethoxam 0.15 g The present bacterial strain 1 × 10¹³ cfuThiamethoxam 0.15 g The present bacterial strain 1 × 10¹⁰ cfuThiamethoxam 0.25 g The present bacterial strain 1 × 10¹³ cfuThiamethoxam 0.25 g

The compartment treated with the composition of the invention shows asynergistic controlling effect, for each combination, compared with thatof the corresponding compartment treated solely with the compound or thepresent bacterial strain.

Test Example 10

In a rotary seed treatment machine (trade name: HEGE11, manufactured byWINTERSTEIGER), corn seeds (variety: yellow dent corn) are treated bysmearing treatment using a wettable powder of the present bacterialstrain as obtained in Formulation Example 11, clothianidin flowableformulation (47.8% flowable formulation, trade name: NipsIt InsideInsecticide, Valent U.S.A. Corporation) or imidacloprid flowableformulation (48.7% flowable formulation, trade name: Gaucho 600, BayerCropScience), so that the soybean seeds thus treated retain the presentbacterial strain, clothianidin or imidacloprid in the amount shown inTable 3.

In a rotary seed treatment machine (trade name: HEGE11, manufactured byWINTERSTEIGER), the corn seeds thus treated with clothianidin orimidacloprid are treated by wet powder coating with a wettable powder ofthe present bacterial strain as obtained in Formulation Example 11, sothat the soybean seeds thus treated retain the present bacterial strainand clothianidin or the present bacterial strain and imidacloprid in theamount shown in Table 3.

A plastic pot is filled with a soil, and then, the corn seeds, whichhave been treated with the present bacterial strain, compound or thepresent bacterial strain+compound as shown in Table 3, are seeded, andcultivation is carried out in a glass greenhouse for given days(“treated compartment”). Newly hatched larvae of Diabrotica virgiferavirgifera are released (10 for each stalk of corn) on the 7th day afterthe seeding, and the number of living Diabrotica virgifera virgifera iscounted on the 10th day after the release.

Similar procedures are conducted using untreated corn seeds, instead ofthe treated corn seeds, as described above for the treated compartment(“untreated compartment”).

Based on the numbers of survivors in the treated compartment and theuntreated compartment, the effect in the treated compartment iscalculated by the following equation.Controlling effect=100×[(number of survivors in untreated compartmentnumber of survivors in treated compartment)/number of survivors inuntreated compartment]

TABLE 3 Bacteria/Compound retained by Retaining Amount Seeds (/Kg seeds)The present bacterial strain 1 × 10⁹ cfu The present bacterial strain 1× 10¹⁰ cfu Clothianidin 0.6 g Clothianidin 1.2 g Imidacloprid 2.0 gImidacloprid 2.5 g The present bacterial strain 1 × 10⁹ cfu Clothianidin0.6 g The present bacterial strain 1 × 10¹⁰ cfu Clothianidin 0.6 g Thepresent bacterial strain 1 × 10⁹ cfu Clothianidin 1.2 g The presentbacterial strain 1 × 10¹⁰ cfu Clothianidin 1.2 g The present bacterialstrain 1 × 10⁹ cfu Imidacloprid 2.0 g The present bacterial strain 1 ×10¹⁰ cfu Imidacloprid 2.0 g The present bacterial strain 1 × 10⁹ cfuImidacloprid 2.5 g The present bacterial strain 1 × 10¹⁰ cfuImidacloprid 2.5 g

The compartment treated with the composition of the invention shows asynergistic controlling effect, for each combination, compared with thatof the corresponding compartment treated solely with the compound or thepresent bacterial strain.

Test Example 11

Spray liquids of a wettable powder of the present bacterial strain asobtained in Formulation Example 11, of clothianidin wettable powder (16%wettable powder, trade name: DANTOTSU wettable powder, Sumitomo ChemicalCompany, Limited), of flupyradifurone flowable formulation (20.0% liquidformulation, trade name: SIVANT0200, Bayer CropScience), of wettableimidacloprid granules (50% wettable granules, trade name: Admirewettable granules, Bayer CropScience), of wettable thiacloprid granules(30% wettable granules, trade name: BARIARD wettable granules, BayerCropScience), and of thiamethoxam water soluble powder (10.0% solublepowder, trade name: Actara Water Soluble Powder, Syngenta Japan) areprepared independently by adjusting their concentrations as shown inTable 4.

The liquid (50 mL) is sprayed to pot planting cucumber plants whereinprimary leaf development has occurred. After air drying, 20 Aphisgossypii are released, and the number of living Aphis gossypii iscounted on the 5th day after the release (“treated compartment”). Also,similar procedures are conducted without spraying the liquid (“untreatedcompartment”).

Based on the numbers of survivors in the treated compartment and theuntreated compartment, the effect in the treated compartment iscalculated by the following equation.Controlling effect=100×[(number of survivors in untreatedcompartment−number of survivors in treated compartment)/number ofsurvivors in untreated compartment]

TABLE 4 Amount of Bacteria/Compound sprayed to Bacteria/Compound inPlants Spray Liquid (/L) The present bacterial strain 1 × 10⁸ cfuClothianidin 0.8 mg Flupyradifurone 1.0 mg Imidacloprid 1.0 mgThiacloprid 1.5 mg Thiamethoxam 0.33 mg  The present bacterial strain 1× 10⁸ cfu Clothianidin 0.8 mg The present bacterial strain 1 × 10⁸ cfuFlupyradifurone 1.0 mg The present bacterial strain 1 × 10⁸ cfuImidacloprid 1.0 mg The present bacterial strain 1 × 10⁸ cfu Thiacloprid1.5 mg The present bacterial strain 1 × 10⁸ cfu Thiamethoxam 0.33 mg 

The compartment treated with the composition of the invention shows asynergistic controlling effect, for each combination, compared with thatof the corresponding compartment treated solely with the compound or thepresent bacterial strain.

Test Example 12

In a rotary seed treatment machine (trade name: HEGE11, manufactured byWINTERSTEIGER), soybean seeds (variety:“Hatayutaka”) were treated bysmearing treatment using flowable formulation of the present bacterialstrain obtained in Formulation Example 14, flupyradifurone liquidformulation (20.0% liquid formulation, trade name: SIVANTO200, BayerCropScience), thiacloprid flowable formulation (40.0% flowableformulation, trade name: Sonido, Bayer CropScience), thiamethoxamflowable formulation (47.6% flowable formulation, trade name: Cruiser5FS, Syngenta Crop Protection LCC), so that the soybean seeds retain thepresent bacterial strain and the compound in the amount shown in Table5.

A plastic pot was filled with a soil, and then, the soybean seeds, whichhave been treated with present bacterial strain, compound or the presentbacterial strain+compound as shown in Table 5, were seeded, andcultivation was carried out in a glass greenhouse for given days(“treated compartment”). Twenty Aphis gossypii were released on the 14thday after the seeding, and the number of living Aphis gossypii wascounted on the 5th day after the release. Similar procedures wereconducted using untreated soybean seeds, instead of the treated soybeanseeds, as described above for the treated compartment (“untreatedcompartment”).

Based on the numbers of survivors in the treated compartment and theuntreated compartment, the effect in the treated compartment wascalculated by the following equation.Controlling effect=100×[(number of survivors in untreatedcompartment−number of survivors in treated compartment)/number ofsurvivors in untreated compartment]

TABLE 5 Retaining Bacteria/Compound Amount Controlling Estimatedretained by Seeds (/Kg seeds) Effect Value* The present bacterial 1 ×10¹⁰ cfu 11.7 — strain The present bacterial 1 × 10¹³ cfu 18.0 — strainFlupyradifurone  0.2 g 65.9 — Flupyradifurone  0.4 g 77.6 — Thiacloprid 0.3 g 56.9 — Thiacloprid 0.45 g 69.0 — Thiamethoxam 0.15 g 70.6 —Thiamethoxam 0.25 g 78.4 — The present bacterial 1 × 10¹⁰ cfu 84.3 69.9strain  0.2 g Flupyradifurone The present bacterial 1 × 10¹³ cfu 89.472.0 strain  0.2 g Flupyradifurone The present bacterial 1 × 10¹⁰ cfu97.2 80.2 strain  0.4 g Flupyradifurone The present bacterial 1 × 10¹³cfu 99.2 81.6 strain  0.4 g Flupyradifurone The present bacterial 1 ×10¹⁰ cfu 81.2 62.0 strain  0.3 g Thiacloprid The present bacterial 1 ×10¹³ cfu 89.0 64.7 strain  0.3 g Thiacloprid The present bacterial 1 ×10¹⁰ cfu 87.0 72.6 strain 0.45 g Thiacloprid The present bacterial 1 ×10¹³ cfu 93.3 74.6 strain 0.45 g Thiacloprid The present bacterial 1 ×10¹⁰ cfu 91.0 74.0 strain 0.15 g Thiamethoxam The present bacterial 1 ×10¹³ cfu 94.1 75.9 strain 0.15 g Thiamethoxam The present bacterial 1 ×10¹⁰ cfu 100 80.9 strain 0.25 g Thiamethoxam The present bacterial 1 ×10¹³ cfu 100 82.3 strain 0.25 g Thiamethoxam *Effect estimated from thecalculation by the Colby's equation

If the effect by the combination of two active ingredients is greaterthan that of the estimated value E, which is calculated by the Colby'sequation as follows, the effect is regarded as synergistic.E=X+Y−X·Y/100wherein,

-   E=Controlling effect when using the mixture of the active    ingredients A and B at the concentrations m and n (amount of the    active ingredient), respectively.-   X=Controlling effect when using the active ingredient A at the    concentration m (amount of the active ingredient).-   Y=Controlling effect when using the active ingredient B at the    concentration n (amount of the active ingredient).

The compartment treated with the composition of the invention showed asynergistic controlling effect, for each combination, compared with thatof the corresponding compartment treated solely with the compound or thepresent bacterial strain.

Test Example 13

In a rotary seed treatment machine (trade name: HEGE11, manufactured byWINTERSTEIGER), corn seeds (variety: yellow dent corn) were treated bysmearing treatment using the wettable powder of the present bacterialstrain obtained in Formulation Example 13 or clothianidin flowableformulation (47.8% flowable formulation, trade name: NipsIt InsideInsecticide, Valent U.S.A. Corporation), or imidacloprid flowableformulation (48.7% flowable formulation, trade name: Gaucho 600, BayerCropScience), so that the corn seeds thus treated retain the presentbacterial strain, clothianidin or imidacloprid in the amount shown inTable 6.

In a rotary seed treatment machine (trade name: HEGE11, manufactured byWINTERSTEIGER), the corn seeds thus treated with clothianidin orimidacloprid were treated by wet powder coating with a wettable powderof the present bacterial strain as obtained in Formulation Example 13,so that the soybean seeds thus treated retain the present bacterialstrain and clothianidin or the present bacterial strain and imidaclopridin the amount shown in Table 6.

A plastic pot was filled with a soil, and then, the corn seeds, whichhave been treated with the present bacterial strain, compound or thepresent bacterial strain+compound as shown in Table 6, were seeded, andcultivation was carried out in a glass greenhouse for given days(“treated compartment”). Newly hatched larvae of Diabrotica virgiferavirgifera are released (10 for each stalk of corn) on the 7th day afterthe seeding, and the number of living Diabrotica virgifera virgifera wascounted on the 10th day after the release. Similar procedures wereconducted using untreated corn seeds, instead of the treated corn seeds,as described above for the treated compartment (“untreatedcompartment”).

Based on the numbers of survivors in the treated compartment and theuntreated compartment, the effect in the treated compartment wascalculated by the following equation.Controlling effect=100×[(number of survivors in untreated compartmentnumber of survivors in treated compartment)/number of survivors inuntreated compartment]

TABLE 6 Retaining Bacteria/Compound Amount Controlling Estimatedretained by Seeds (/Kg seeds) Effect Value* The present bacterial 1 ×10⁹ cfu 11.4 — strain The present bacterial 1 × 10¹⁰ cfu 21.4 — strainClothianidin 0.6 g 41.4 — Clothianidin 1.2 g 64.3 — Imidacloprid 2.0 g51.4 — Imidacloprid 2.5 g 62.8 — The present bacterial 1 × 10⁹ cfu 58.648.1 strain 0.6 g Clothianidin The present bacterial 1 × 10¹⁰ cfu 70.053.9 strain 0.6 g Clothianidin The present bacterial 1 × 10⁹ cfu 84.368.4 strain 1.2 g Clothianidin The present bacterial 1 × 10¹⁰ cfu 94.371.9 strain 1.2 g Clothianidin The present bacterial 1 × 10⁹ cfu 72.856.9 strain 2.0 g Imidacloprid The present bacterial 1 × 10¹⁰ cfu 81.461.8 strain 2.0 g Imidacloprid The present bacterial 1 × 10⁹ cfu 90.067.0 strain 2.5 g Imidacloprid The present bacterial 1 × 10¹⁰ cfu 92.970.8 strain 2.5 g Imidacloprid *Control value estimated from thecalculation by the Colby's equation

The compartment treated with the composition of the invention showed asynergistic controlling effect, for each combination, compared with thatof the corresponding compartment treated solely with the compound or thepresent bacterial strain.

The invention claimed is:
 1. A composition for controlling pests comprising a synergistic effective amount of Bacillus strain APM-1 deposited under ATCC Accession No. PTA-4838 and one or more nicotinic acetylcholine receptor agonist compound selected from the group consisting of clothianidin, flupyradifurone, imidacloprid, thiacloprid and thiamethoxam, wherein the composition comprises the nicotinic acetylcholine receptor agonist compound in the amount of 10⁻⁴ to 10² g per 10¹⁰ cfu of Bacillus strain APM-1, and wherein the pests are Hemipteran pests, Lepidopteran pests, Thysanopteran pests, Dipteran pests, or Coleopteran pests.
 2. A plant seed or a vegetative propagation organ comprising a synergistic effective amount of Bacillus strain APM-1 deposited under ATCC Accession No. PTA-4838 and one or more nicotinic acetylcholine receptor agonist compound selected from the group consisting of clothianidin, flupyradifurone, imidacloprid, thiacloprid and thiamethoxam, wherein the plant seed or the vegetative propagation organ comprises 10⁶ to 10¹³ cfu of Bacillus strain APM-1 and 0.01 to 10 g of the nicotinic acetylcholine receptor agonist compound, per 1 kg of the seed or vegetative propagation organ.
 3. A method for controlling pests, comprising a step of applying a synergistic effective amount of Bacillus strain APM 1 deposited under ATCC Accession No. PTA-4838 and one or more nicotinic acetylcholine receptor agonist compound selected from the group consisting of clothianidin, flupyradifurone, imidacloprid, thiacloprid and thiamethoxam, to a plant or a plant cultivation site, wherein the nicotinic acetylcholine receptor agonist compound is applied in the amount of 10⁻⁴ to 10² g per 10¹⁰ cfu of Bacillus strain APM-1, and wherein the pests are Hemipteran pests, Lepidopteran pests, Thysanopteran pests, Dipteran pests, or Coleopteran pests.
 4. The method for controlling pests according to claim 3 wherein the plant is a genetically modified plant.
 5. A method for controlling pests, comprising a step of applying a synergistic effective amount of Bacillus strain APM 1 deposited under ATCC Accession No. PTA-4838 and one or more nicotinic acetylcholine receptor agonist compound selected from the group consisting of clothianidin, flupyradifurone, imidacloprid, thiacloprid and thiamethoxam, to pests or a place where pests are liable to inhabit, wherein the nicotinic acetylcholine receptor agonist compound is applied in the amount of 10⁻⁴ to 10² g per 10¹⁰ cfu of Bacillus strain APM-1, and wherein the pests are Hemipteran pests, Lepidopteran pests, Thysanopteran pests, Dipteran pests, or Coleopteran pests. 